Fatty acids, coco, iso-Bu esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1953

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable published study, basic data given.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 20 male and female rats were treated during 10 weeks with the test item at a concentration of 6.25% in diet and were then mated. F1 animals were also treated post weaning and sacrificed at day 21 post weaning.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) 5-6 weeks
- Weight at study initiation: Control group: (F1) Males: 44 ± 8 g; Females: 43 ± 9 g; Treatment group: (F1) Males: 39 ± 6 g; Females: 37 ± 6 g

Route of administration:

oral: feed

Vehicle:

not specified

Details on mating procedure:

- After successful mating each pregnant female was caged: Pregnant females were transferred to individual breeding cages.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

(P) Males and Females: 10 weeks before mating
(F1) 24 males and 24 females were chosen at random and 21 days post partum fed the same diet

Frequency of treatment:

Parental animals:
Daily for 10 weeks

F1 animals:
Daily for 21 days post partum

Details on study schedule:

- Parental animals were fed diets containing 6.25% test substance for 10 weeks and mated. A control group of 12 male and 12 female animals of the same age were fed the basal ratio for 10 weeks and mated.
- Litters were weaned 21 days post partum.
- 24 male and 24 female litters were chosen at random and 21 days post partum fed the same diet as had been ingested by their parents.

Remarks:

Doses / Concentrations:
6.25%
Basis:
nominal in diet
corresponding to about 6000 mg/kg bw/day

No. of animals per sex per dose:

Parental animals:
Control group: 12 males and 12 females
Treatment group: 20 males and 20 females

F1 animals:
Treatment group: 24 males and 24 females

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

Examination of parental animals was focused on fertility.

Litter observations:

The following parameters were examined in [F1] offspring: number and sex of pups, litter size, survival of offspring, body weight (daily), weight gain.

Postmortem examinations (parental animals):

Not specified.

Postmortem examinations (offspring):

The F1 offspring were sacrificed 21 days after weaning and and were subjected to postmortem examinations.

Reproductive performance:

no effects observed

No adverse effects were noted between control and treatment groups.
No adverse effects were noted with respect to fertility.

Dose descriptor:

NOAEL

Effect level:

ca. 6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted with respect to fertility.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

non-adverse

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

No adverse effects were noted between control and treatment groups.
No adverse effects were found with respect to litter size of offspring. Only the growth was significantly retarded during the preweaning and postweaning periods.
No gross lesions were found among the animals at sacrifice at the end of the 21-day postweaning period.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Table 1: Summary of effects of butyl stearate on reproduction and growth of offspring in the rat

Substance in diet, %	No. of females		No. of rats per litter				Mean weight at weaning ± deviation [g]		Mean weight gain 21 days after weaning ± deviation [g]
	In group	Bearing young	At birth		At weaning (21 days)
			mean	range	mean	range	male	female	male	female
None	12	12	9.6	6-12	8.8	6-11	44±8	43±9	110±15	96±10
6.25	20	20	10.5	3-11	9.3	3-11	39±6*	37±6 t	101±10*	86±7 **

* P = > 0.01 < 0.05

** P = < 0.01

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) and the source substance FATTY ACIDS, C16-18, ISOBUTYL ESTERS (CAS 85865-69-6) are both Short Chain Alcohol Esters (SCAE C2-C8) composed by a fatty acid (C16-C18) and a C4 alcohol (isobutanol).
The source and the target substance show therefore the same reactive groups and a similar composition. A read-across to the source is therefore justified.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both target and source substances are fatty acid esters produced by chemical reaction of an alcohol (isobutanol) with organic acids (e. g. stearic acid) in the presence of an acid catalyst. The esterification reaction is started by a transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The carboxylic acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to a carbonyl carbon of acid. An intermediate product is formed. This intermediate product loses a water molecule and proton to give an ester. Monoesters are the final product of esterification.

3. ANALOGUE APPROACH JUSTIFICATION
Since both target and source substances are fatty acid esters produced by chemical reaction of an alcohol (isobutanol) with an organic acid and therefore share similar/overlapping structural features and functional groups, it is justified to use a read across approach. The source substance has been registered already and its toxicity to reproduction has been investigated using a grouping of substance and read across approach. The reproductive/developmental toxicity of the source substance, Fatty acids, C16-18, Isobutyl Esters (CAS# 85865-69-6), was tested in two tests. The studies did not show treatment-related effects up to the highest tested dose level. Thus, no hazard for reproduction toxicity was identified.
All available data investigating the toxicity to reproduction indicate that members of the category Fatty acid C2-8 esters have no reproduction toxicity effects and classification according to EU classification criteria for reproductive toxicity is not required.
The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Dose descriptor:

NOAEL

Remarks:

parental fertility

Effect level:

ca. 6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were noted with respect to fertility.

Dose descriptor:

NOAEL

Remarks:

offspring development

Generation:

F1

Effect level:

ca. 6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed with respect to litter size and survival of offspring.

Reproductive effects observed:

not specified

Conclusions:

All available data investigating the toxicity to reproduction indicate that members of the category Fatty acid C2-8 esters have no reproduction toxicity effects and classification according to EU classification criteria for reproductive toxicity is not required.
The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .

Executive summary:

The source substance has been registered already and its toxicity to reproduction has been investigated using a grouping of substance and read across approach. The reproductive/developmental toxicity of the source substance, Fatty acids, C16-18, Isobutyl Esters (CAS# 85865-69-6), was tested in two tests. The studies did not show treatment-related effects up to the highest tested dose level. Thus, no hazard for reproduction toxicity was identified.

All available data investigating the toxicity to reproduction indicate that members of the category Fatty acid C2-8 esters have no reproduction toxicity effects and classification according to EU classification criteria for reproductive toxicity is not required.

The same behaviour is predicted for the target substance FATTY ACIDS, COCO, ISO-BU ESTERS (CAS 91697-43-7) .

Reference 3

Endpoint:

toxicity to reproduction

Remarks:

other: extended 90 day feeding study with fertility parameters

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented published GLP guideline study.

Qualifier:

according to guideline

Guideline:

other: 1993 FDA draft "Redbook II" guidelines (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food).

Principles of method if other than guideline:

The purpose of this study was to determine the safety of ethyl oleate (EO) in a 91-day feeding study in Sprague-Dawley rats.
Additionally to the repeated dose toxicity, oestrus cycle and sperm parameters were analyzed.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory
- Strain as given in publication: Sprague-Dawley rats [Crl:CD(SD)IGS BR]
- Age at study initiation: approx. 5-6 weeks
- Weight at study initiation: approx. 150–175 g
- Fasting period before study: one night prior to blood collections
- Housing: individually in stainless steel cages
- Diet: AIN-93G diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
Temperature and humidity were controlled throughout the duration of the study.

Route of administration:

oral: feed

Vehicle:

other: diet containing high oleic safflower oil (HOSO)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated into the diet. For this purpose, the AIN-93G diet was modified to allow incorporation of an additional 10% of test fat (either EO, HOSO, or a combination of the two). The modification involved decreasing overall carbohydrate concentration to allow the incorporation of an additional 10% of test fat without diluting out other nutrients.
The test diets were prepared based on the addition of the EO oil (i.e., the high-dose diets contained 10% of the EO oil), and were not adjusted based on the actual concentration of the EO molecule.

VEHICLE
- Source: High oleic safflower oil (HOSO) was obtained from Columbus Foods Company, Chicago, Illinois.

Details on mating procedure:

no matings performed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Data on test material stability, homogeneity, and diet concentrations showed that the test material was stable over the course of the study, and diet concentrations were homogeneous and within 10% of target (data not shown).

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily ad libitum feeding

Remarks:

Doses / Concentrations:
2.0, 3.9, 6.1 g/kg bw/day
Basis:
other: females, mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % in feed

Remarks:

Doses / Concentrations:
1.8, 3.6, 5.5 g/kg bw/day
Basis:
other: males, mean dose value as calculated from the actual body weight and food consumption data and dietary target concentration of 3.3, 6.7 and 10 % in feed

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

see 7.5.1: Bookstaff, 2004, rat, 90d oral, RL2

Oestrous cyclicity (parental animals):

Beginning on the first day of Week 11 and continuing for 21 consecutive days, all females had daily vaginal smears prepared and examined to evaluate the stage of the estrous cycle.

Sperm parameters (parental animals):

Parameters examined: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology.
At the terminal sacrifice, males were evaluated for sperm viability.
- For motility and morphology assessment, the right vas deferens was excised and immediately placed in a petri dish containing 10 mL of a 1% bovine serum albumin dissolved in phosphate buffered saline. The solution was prewarmed to approximately 38°C. A 3- to 4-min period (minimum to maximum period, respectively) was allowed for the sperm to swim out. Following the swim-out period, a sample of sperm was collected using a 100-micron-deep cannula and immediately loaded into a prewarmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields/animal were selected and stored as digital images. These images were analyzed for percent motility.
- Sperm morphology was assessed with two slides of sperm stained with eosin for each male. A minimum of 200 sperm cells was evaluated.
- For sperm count, the right epididymis was removed and divided in half by cross sectioning through the middle. The tail end caudal section was placed on dry ice, and stored frozen at approximately -60 to -80°C until analysis for total sperm count.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: all animals were subjected to gross pathological examination

ORGAN WEIGHTS: the following organs were weighed (paired organs weighed together). Organ-to-body weight percentages and organ-to-brain weight ratios were calculated: adrenal (2), pituitary gland, brain, prostate, epididymides (2), spleen, heart, testis (2), kidney (2), thymus, liver, thyroid with parathyroid, ovary (2), uterus.

HISTOPATHOLOGY: histopathology was done in control and high dose group animals.
The following tissues (when present) from each animal, with the exception of testes, were preserved in 10% neutral-buffered formalin and slides prepared for histopathological examination. Testes were preserved in Bouins fixative.
Adrenal (2)
Aorta
Brain (cerebrum, cerebellum, and medulla)
Cecum
Cervix
Colon [proximal and distal (2)]
Duodenum
Epididymis (2)
Esophagus
Eye (2)
Femur with bone marrow (articular surface of
the distal end)
Harderian gland
Heart
Ileum (including Peyers patch)
Jejunum
Kidney (2)
Lacrimal gland (exorbital)
Liver
Lung with mainstem bronchi
Lymph node (mandibular and mesenteric)
Mammary gland (females)
Nasal turbinates
Ovary (2)
Pancreas
Pituitary gland
Prostate
Rectum
Salivary gland [mandibular (2)]
Sciatic nerve
Seminal vesicle (2)
Skeletal muscle (thigh)
Skin
Spinal cord (cervical, thracic, lumbar)
Spleen
Sternum with bone marrow
Stomach (nonglandular and glandular)
Testis [preserved in Bouins fixative for
sacrificed animals (2)]
Thymus
Thyroid with parathyroid
Tissues with macroscopic changes or alterations
(i.e., gross lesions)
Tongue
Trachea
Urinary bladder
Uterus with uterine horns
Vagina
Zymbals gland

Statistics:

Control versus treated group comparisons (Groups 2 through 4 versus Group 1) were evaluated at the 5.0%, two-tailed probability level. Data for each sex were analyzed separately. If Levene's test for variance homogeneity was not significant (p > 0.05), one-way analysis of variance (ANOVA) was performed on the observed values. If Levene's test was significant (p < 0.05), ANOVA was done on the rank transformed data. Post-hoc Dunnett's t-test was used for control versus treated group mean comparison, incorporating transformations when necessary.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: decreased food intake due to lower palatability

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no treatment-related effects on reproductive capacity as evaluated by female estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no treatment-related effects on reproductive capacity as evaluated by sperm motility, sperm count, or sperm morphology.

HISTOPATHOLOGY: NON-NEOPLASTIC
Referring to reproduction organs, no histopathological changes of any relevance were reported.

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

5 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: oestrus cyclus in females, sperm characterization in males and histologic examinations (incl. Epididymides, Mammary gland, Ovaries, Prostate, Seminal vesicles, Testes, Thyroid with parathyroid, Uterus with uterine horns and Vagina)

Remarks on result:

other: Mating of the parental animals was not performed.

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

6 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

6 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available